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The soybean meal was used as the raw material to prepare soy protein isolate (SPI) with high emulsification activity (EA) by the method of microwave-assisted phosphorylation. A response model was established by the central composite rotatable design, with concentration of SPI, ratio of sodium tripolyphosphate (STP), microwave power, time of reaction as four variables and the emulsification activity (EA) as the evaluation index. Response surface analysis revealed that the optimized conditions of preparation of soybean protein isolate with high EA by microwave-assisted phosphorylation were as follows: concentration of SPI 10%, ratio of STP 16%, microwave power 480W and time of reaction 4min. The response model was valid in analyzing and predicting the extent of EA due to its higher fitting degree and less experimental error. Under the optimal conditions, the EA reached 66.8 and the emulsification stability (ES) reached 29.80min. Compared with the previously unmodified SPI, the EA and the ES were increased by 134.4% and 61.6%, respectively.